The objective of this randomized, crossover, pilot study was to assess the effect of aqua-acupuncture (aqua-AP) at An-shen acupoint on heart rate (HR) in healthy dogs. Thirteen dogs were randomized to receive aqua-AP at An-shen acupoint using solutions of normal saline, Traumeel®, and cyanocobalamin (vitamin B12) with a minimum of one-week washout period between crossovers. An ambulatory electrocardiography (Holter) was recorded for 24 hours before and after each aqua-AP to assess the changes in 24-hour mean HR, maximum HR, and minimum HR. No dogs experienced any adverse effects from aqua-AP treatment. After aqua-AP, 91% of dogs with vitamin B12, 60% with Traumeel®, and 42% with normal saline experienced a decrease in 24-hour mean HR. The mean (with 95% confidence interval) decreases in 24-hour mean HR (bpm) from baseline was 4.18 (1.8 to 6.5) with vitamin B12, 3.9 (-3.7 to 3.1) with Traumeel®, and -0.25 (-7.7 to -1.1) with normal saline. The changes in maximum and minimum HR were variable with wider confidence estimates around the mean change in the respective HR’s for any aqua-AP solution. The results of this pilot study show that aqua-AP with vitamin B12 provided the greatest decrease in 24-hour mean HR in healthy dogs. Further confirmatory studies are warranted to assess the effect of aqua-AP on HR in clinical conditions that warrant HR reduction.